www.elsevier.comrlocateranireprosci
Genetic influences on reproduction of female red
ž
/
deer Cer
Õ
us elaphus
ž /
2 Seasonal and genetic effects on the
superovulatory response to exogenous FSH
G.W. Asher
), K.T. O’Neill, I.C. Scott, B.G. Mockett, A.J. Pearse
AgResearch, InÕermay Agricultural Centre, Puddle Alley, PriÕate Bag 50034, Mosgiel, New Zealand
Received 11 August 1999; received in revised form 7 December 1999; accepted 7 December 1999
Abstract
This study evaluated the influences of seasons and genotype on the superovulatory response to
Ž .
a standardised oFSH regimen in red deer CerÕus elaphus scoticus and its hybrids with either
Ž . Ž . Ž . Ž .
wapiti C.e. nelsoni or Pere David’s PD deer Elaphurus da` Õidianus . Adult red deer ns9 ,
Ž . Ž
F hybrid wapiti1 =red deer ns6 , and maternal backcross hybrid PD=red deer i.e., 1 4 PD
.
hybrid; ns9 were kept together in the presence of a vasectomised stag for 13 months. At 6 weekly intervals, all hinds received a standardised treatment regimen used routinely to induce a superovulatory response in red deer hinds, with 10 consecutive treatments spanning an entire year. This involved synchronisation with intravaginal progesterone devices and delivery of multiple
Ž .
injections of oFSH equivalent to 72 units NIH-FSH-S . Laparoscopy to assess ovarian response1 was performed 6–7 days after the removal of the devices. Both season and genotype had
Ž . Ž . Ž .
significant effects on ovulation rate OR and total follicular stimulation TFS P-0.05 . For all
Ž .
the three genotypes, ovarian responses were highest from March to November breeding season and lowest in the period from December to January, inclusive. Mean OR for red deer hinds ranged from 3.7 to 1.8 during the breeding season, with no observable trend. All red deer hinds were anovulatory during December and January. A similar pattern occurred for 1 4 PD hybrids, although mean OR during the breeding seasons were twofold lower than for the red deer. For F1 wapiti hybrids, the first two treatments in March and April resulted in the highest mean OR
Ž .
observed 15.6 and 11.7, respectively . Thereafter, mean values ranged between 6.3 and 4.7 for the remainder of the breeding season. Furthermore, mean OR of 3.0 and 0.5 were recorded in
)Corresponding author. Tel.:q64-3489-3809; fax:q64-3489-3739.
0378-4320r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved.
Ž .
( ) G.W. Asher et al.rAnimal Reproduction Science 59 2000 61–70 62
December and January, respectively. For the red deer and F wapiti hybrids, between-hind1 variation in OR was not randomly distributed across the treatment dates, indicating that the individuals varied significantly in their ability to respond to oFSH, at least within a given season. In conclusion, the study has shown that relative to red deer, F wapiti hybrid hinds exhibit a1 higher sensitivity to oFSH, whereas 1 4 PD hybrid hinds have a lower sensitivity. However, individual variation within genotype was very marked. A seasonal effect was apparent for all genotypes, although some F wapiti hybrid hinds exhibited ovulatory responses throughout the1 year.q2000 Elsevier Science B.V. All rights reserved.
Keywords: Red deer; Genetics; Seasonal; Dynamics; Superovulation
1. Introduction
Ž . Ž
Multiple ovulation-embryo transfer MOET protocols for red deer CerÕus elaphus
.
scoticus have been established commercially over the last decade, particularly within
the New Zealand deer farming industry. However, inconsistency of the superovulatory
Ž
responses of donor hinds has been a general feature of all programmes Fennessy et al.,
.
1994; Asher et al., 1995 . Seasonality and donor genotype are considered to be two major factors that may contribute to the wide variation of ovarian response to exogenous gonadotrophins.
The highly seasonal nature of luteal cyclicity of red deer hinds has been well
Ž .
documented Guinness et al., 1971; Duckworth and Barrell, 1992; Asher et al., 2000 .
Ž .
The ‘‘rut’’ i.e., the season of intense sexual activity occurs during the early part of the hinds’ potential breeding seasons. It is also the time when most of the MOET programmes are performed. However, seasonal constraints on an optimal calving season in early summer often necessitate the instigation of MOET programmes prior to the rut
ŽFennessy et al., 1994 , at a time when a proportion of hinds may be exhibiting.
anovulation and luteal absence. It seems likely that this would lead to highly variable superovulation responses. Conversely, few studies have considered ovarian responses to exogenous gonadotrophins in the period after the rut. This has relevance to the programmes of repeated embryo collections from elite donors.
Previous studies have demonstrated genetic influences on the timing and duration of
Ž
the breeding season of ‘‘red deer’’ C. e. scoticus vs. subspecies and species hybrids;
.
Asher et al., 2000 , which may influence the superovulatory responses early in the breeding season. There are also putative direct genetic effects on ovarian responses to exogenous gonadotrophins. Anecdotal evidence indicates variable sensitivity to
exoge-Ž
nous FSH amongst various genotypes, with the deer of eastern European origin C. e.
. Ž
hippelaphus being reputably more reliably responsive to treatment N. Beatson,
per-. Ž
sonal communication , and the North American wapiti C. e. nelsoni, C. e. rooseÕelti,
.
C. e. manitobensis, etc. characteristically exhibiting very poor responses to treatment ŽBringans, 1987, 1989 ..
The objective of the present study was to evaluate the influences of season and
Ž .
2. Materials and methods
2.1. Ethical considerations
These studies were undertaken with the approval of the AgResearch Invermay
Ž
Animal Ethics Committee, as required in New Zealand by the Animal Welfare Codes of
.
Ethical Conduct Act 1987. All procedures were conducted by fully trained staff from
Ž
the Invermay Agricultural Centre and in accredited facilities NZQA accreditation
.
scheme .
2.2. Animals and management
Ž .
This study was conducted in the year subsequent i.e., 1996 to that described by
Ž . Ž
Asher et al. 2000 using the same animals. The group was comprised of 25 adult 5–11
. Ž . Ž
years old parous hinds of the three distinct genotypes : i Scottish red deer C. e.
. Ž . Ž . Ž .
scoticus; ns9 ; ii F hybrid wapiti C. e. nelsoni1 =red deer C. e. scoticus; ns6 ;
Ž . Ž . Ž
and iii maternal backcross hybrid PD deer E. daÕidianus; 25% =red deer C. e.
.
scoticus; 75%; ns9 . However, following the deaths of one red deer hind and one PD
Ž .
deer hybrid hind from malignant catarrhal fever MCF in September 1996, replacement individuals of corresponding genotype were included in the study at this time. The hinds were grazed on predominantly ryegrassrclover pasture as a single group in the continuous presence of a vasectomised red deer stag throughout the duration of the trial from February 1996 to March 1997.
2.3. SuperoÕulation treatment
At intervals of exactly 6 weeks starting in February 1996, all hinds received a standardised treatment regimen used routinely to induce a superovulatory response in
Ž .
red deer hinds Fennessy et al., 1994 . This treatment consisted of a 12-day placement of
Ž
an intravaginal progesterone-releasing device Easi-breed CIDR type G; InterAg,
Hamil-.
ton, NZ , with device replacement on the eighth day. A series of eight consecutive i.m.
Ž
injections of 0.05 units ovine FSH Ovagen; Immuno-Chemical Products, Auckland,
.
NZ; 0.04 units total dosage per animal equivalent to 72 units NIH-FSH-S1 was delivered approximately every 12 h from the device replacement to 12 h after the final device removal. The final injection also contained 250 i.u. equine chorionic
go-Ž .
nadotrophin eCG; Folligon; Intervet, Lane Cove, NSW, Australia . Additional treatment to ensure complete regression of all luteal tissues prior to the next superovulatory
Ž
treatment involved the i.m. injection of 500 mg cloprostenol 2 ml Estrumate; Coopers
.
Animal Health NZ, Upper Hutt, NZ 14 days after the removal of the CIDR device.
2.4. Assessment of oÕarian response
All hinds underwent laparascopic ovarian examination 6–7 days after the device
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( ) G.W. Asher et al.rAnimal Reproduction Science 59 2000 61–70 64
y1 y1 Ž
xylazine hydrochloride, 3.2 mg ml azaperone, and 0.4 mg ml fentanyl citrate 0.02
y1 .
ml kg liveweight Fentazin; Parnell Laboratories, Auckland, NZ . Uterine turgidity
Ž
was reduced by an i.v. injection of 0.5 mg clenbuterol hydrochloride 5 ml Planipart;
Ž . .
Boehringer Ingelheim NZ , Auckland 2–3 min prior to laparoscopy. Each ovary was examined for the presence and the number of corpora lutea, luteinised follicles, and non-luteinised follicles with a diameter greater than or equal to 8 mm. Upon the completion of the ovarian examination, the hinds were given an i.v. injection of 50 mg
Ž .
yohimbine hydrochloride 5 ml Recervyl; Aspiring Animal Services, Wanaka, NZ to reverse the effects of anaesthesia.
2.5. Measurement of oFSH antibody in plasma
All hinds in the trial were blood-sampled by jugular venepuncture at the start, middle, and end of the study. Plasma samples were diluted 1:100 in pH 7.0 phosphate buffered
Ž . Ž .
saline containing 0.1% BSA PBS BSA . Diluted samples 100 ml were incubated
overnight with 100 ml pre-precipitated sheep anti-rabbit second antibody, the samples
were then incubated for 1 h and then centrifuged at 3200 rpm for 35 min after the addition of 1.0 ml of 4% WrV PEG 6000. Samples were decanted and counted for 60 s. The oFSH binding activity of the plasma was determined from the ratio of bound to the total counts following correction for non-specific binding controls.
2.6. Statistical analyses
Ž .
Ovulation rate OR was assessed as the number of corpora lutea and the total
Ž .
follicular stimulation TFS was calculated as the sum of corpora lutea, luteinised
Ž qx.
follicles, and large non-luteinised follicles. OR and TFS data were log 1
trans-Ž . Ž
formed and analysed by residual maximum likelihood REML Patterson and
Thomp-.
son, 1971 with individuals as a random effect, and genotype, date, and their interaction as fixed effects. The assumption of individual randomness in ovarian response over time was evaluated for each genotype independently by the non-parametric means test
ŽFriedman’s Test based on ovulation rate rankings. This evaluation was conducted for.
the first five consecutive treatments only due to missing values for some animals at subsequent dates.
3. Results
Loss of CIDR devices occurred on four occasions at various times during the study. The data at these observations were excluded from the study. There were highly significant effects of both the season and genotype on ovarian response to oFSH
Ž . Ž .
treatment P-0.05; Table 1 and Fig. 1 with minimal interaction P)0.10 . For all of
Ž .
the three genotypes, ovarian responses OR and TFS were greatest from March to
Ž . Ž
November hereafter referred to as the breeding season , and least even non-existent for
. Ž .
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Table 1
Ž .
Summary of the overall ovarian response to FSH treatment by genotype
a b
Genotype Observation Mean OR Mean TFS ORrTFS Nil-responses OR?3 CL OR?10 CL
Ž .n ŽS.E.M.. ŽS.E.M.. ratio Ž . Ž% i.e., anovulation. Ž .% Ž .%
Ž . Ž .
Red 89 2.15 0.42 3.21 0.51 0.67 58.4 23.6 8.9
Ž . Ž .
1 4 PD hybrid 77 1.17 0.18 1.61 0.23 0.73 45.5 18.2 0
Ž . Ž .
Wapiti hybrid 59 6.32 0.92 7.25 0.96 0.87 35.6 55.9 32.2
a Ž .
ORsovulation rate number of CL .
b
( ) G.W. Asher et al.rAnimal Reproduction Science 59 2000 61–70 66
Ž . Ž . Ž .
Fig. 1. Mean qS.E.M. ovulation rates shaded and total follicular stimulation open at each treatment
Ž . Ž . Ž .
period for a red deer, b 1 4 PD hybrids, and c F wapiti hybrids.1
1.8 in August, with no indication of any trends associated with time during the breeding season. However, all red deer hinds were anovulatory during December and January, and exhibited little in the way of any follicular stimulation. A similar seasonal pattern was observed for 1 4 PD hybrids, although the overall mean ovulation rates during the breeding season, ranging from 2.3 in September to 0.5 in November, were consistently lower than for red deer. For F wapiti hybrids, the first two treatment regimens in March1
Ž
and April 1996 were associated with the highest mean ovulation rates 15.6 and 11.7,
.
respectively observed at any time during the trial. Thereafter, mean values during the
Ž .
breeding season and including March 1997 ranged between 6.3 and 4.7. In contrast to red deer and 1 4 PD hybrids, the F wapiti hybrids did not all become anovulatory in1
December and January, with observed mean ovulation rates of 3.0 and 0.5, respectively.
Ž .
Ž .
than for the other genotypes P-0.05 . It is noteworthy that while ovulations were recorded for all of the three genotypes in late September and early November,;60% of the CL observed showed signs of premature regression.
Genotype effects were further highlighted by the overall statistics on the proportion
Ž .
of non-responders i.e., hinds anovulatory following treatment and the proportion of
Ž . Ž
hinds exhibiting either G3 CL i.e., a ‘‘superovulation’’ response or G10 CL i.e., a
. Ž .
‘‘mega-ovulation’’ response Table 1 . For the data pooled for the entire study, F1
Ž .
wapiti hybrids had the lowest number of non-responders 35.6% and most responders
Ž55.9% and 32.2%, respectively, for G3 and G10 ovulations . Red deer exhibited the.
Ž . Ž
highest non-responder rate 58.4% , but 1 4 PD hybrids had the least responders 18.2%
. Ž .
and 0%, respectively, for G3 and G10 ovulations Table 1 . The overall ratio of OR
Ž .
to TFS tended to favour the F wapiti hybrids 0.87 , with the red deer being lowest1 Ž0.67. ŽP-0.05 , indicating a greater tendency for the stimulation of non-ovulating.
follicles.
Ž
The evaluation of individual hind’s ovulatory responses to treatment over time first
.
five consecutive treatments revealed a highly significant non-random pattern for
Ž .
individual red deer Friedman’s Test T2s6.44, P-0.01 and F1 wapiti hybrids
ŽT2s4.65, P-0.01 , but not for 1 4 PD hybrids T. Ž 2s0.23, NS . For the former two.
genotypes, one or two individuals consistently exhibited the highest ovulation rates. Conversely, one red deer hind failed to exhibit any ovulatory responses throughout the trial.
Analysis of plasma samples collected from hinds during the trial failed to reveal any immunological reactivity to oFSH at all, with all values falling below the non-specific binding threshold.
4. Discussion
While the present study has demonstrated marked genotype and seasonality effects on the ovulatory response to oFSH in red deer ‘‘type’’ hinds, the apparent genetic effects are, perhaps, the more interesting given the magnitude of difference observed. The synchronisationrsuperovulation regimen used throughout the study is identical to that used routinely by our group for MOET programmes using elite red deer hinds as donors
ŽFennessy et al., 1994 and has consistently yielded mean ovulation rates of 5–9 CL. Ž
across 4–5 years of application Asher et al., 1995; G.W. Asher and I.C. Scott,
.
unpublished data . It is therefore apparent that the mean ovulation rates for red deer
Ž .
hinds observed in the present study i.e., 1.8–3.7 during the breeding season were generally lower than anticipated. Also of concern was the high proportion of
non-re-Ž . Ž
sponders 58.4% . This may reflect the use of hinds that were not genetically elite i.e.,
.
top 5% of herd for given traits but were rather drawn from the wider breeding herd
Žand, for that matter, more likely to have been used as recipients than donors in a MOET
.
( ) G.W. Asher et al.rAnimal Reproduction Science 59 2000 61–70 68
Ž .
in red deer I.C. Scott, unpublished data . However, the same batch was used throughout the present study. The red deer hinds in the present study do provide a benchmark against which to assess the responses of other related genotypes.
The 1 4 PD hybrid hinds exhibited low OR and TFS responses, as well as a low proportion of superovulatory responses. Mean ovulation rate at any treatment during the breeding season was only about 50% of that observed for red deer. Similarly, Argo et al.
Ž1994 , using an almost identical protocol, found that pure PD deer hinds exhibited a.
Ž .
lower mean superovulation response than red deer hinds i.e., 3.8 vs. 10.8 . Collectively, the two studies indicate reduced sensitivity of the PD genotype to oFSH. However, other factors need to be considered. First, an important consideration of the efficacy of oFSH in pure and hybrid PD genotypes is body size and fatness. Pure PD hinds are about 70%
Ž .
heavier than red deer hinds Argo et al., 1994 . Likewise, the 1 4 PD hybrid hinds in the present study were ;25% heavier then their red deer herdmates, and were also considerably fatter. Therefore, there may well be overall effects of oFSH dose: mass ratios, as well as fat absorptionrimmobilisation of injected oFSH. Second, the overall reproductive performance of 1 4 PD hybrid hinds on the Invermay deer farm has been relatively poor in comparison to red deer. When mated to red deer stags, the hybrid
Ž .
hinds have consistently exhibited higher rates of bareness 30% vs. -5% , foetal loss
Ž8% vs. 2% , and periparturient neonate mortality 25% vs. 5% than similarly mated red. Ž .
Ž .
deer hinds Goosen, 1997 . It is possible, therefore, that the low superovulatory response of the 1 4 PD hybrid hinds may partly reflect overall reproductive dysfunctionality that
Ž .
may relate to the wide genetic distance between parental genotypes Tate et al., 1997 . A surprising result of the study was the comparatively high ovarian response observed for F wapiti hybrid hinds, particularly at the first two treatment sessions. This1 was further reflected in the lowest rate of non-responders and highest rate of superovula-tion. The overall mean OR of the F wapiti hybrids was three times higher than for the1
Ž
red deer. This result is surprising because pure North American Wapiti i.e., the paternal
. Ž .
genotype are known to respond poorly to oFSH preparations Bringans, 1987, 1989 rendering MOET programmes with low chances of success. Also, as the F hybrids used1
Ž .
in the present study weighed ;150 kg cf. 95 kg for the red deer , the dose rate of oFSH per unit liveweight for the hybrids was 30–40% lower than that for the red deer. This strongly indicates that hybrid genotypes have a higher sensitivity to oFSH, despite an apparent converse in pure wapiti.
Repeated treatment of hinds during a 12-month period demonstrated a marked seasonal component to ovarian responsiveness. The seasonality observed loosely
mir-Ž
rored natural luteal cyclicity observed in a previous study using the same animals Asher
.
et al., 2000 . While there were some subtle differences in the seasonal onset of the breeding season between the three genotypes, luteal cyclicity generally occurred from
Ž
early April to late September, followed by a 5-month period of anoestrus Asher et al.,
.
1999 . Ovulation responses to oFSH were generally observed from March to November, inclusive, for all of the three genotypes, with some F wapiti hybrid hinds also ovulating1 in December and January. The induction of superovulation can be successfully achieved
Ž .
indicate that luteal insufficiency during the transition phase into anoestrum may seri-ously limit the application of MOET at this time of year.
There is a considerable variation between individuals in ovarian response to a standardised oFSH regimen within a given genotype. The study has also demonstrated that this variation was not random across consecutive treatments within the breeding season. Some hinds were consistently high responders while others were consistently low responders. It is not known whether these individual characteristics are inherent or
Ž .
acquired i.e., genetic or environmental . However, the consequences on reproductive productivity of donor hinds cannot be ignored, with the numbers of progeny yielded from MOET programmes being strongly biased towards high responding hinds. Some elite genetic material may be lost through ovulatory failure. Such considerations are well known, given the variation in superovulatory responses observed in several red deer
Ž .
studies Fennessy et al., 1989, 1994; Dixon et al., 1991; Asher et al., 1995 .
From a practical perspective, it has not been a common practice in New Zealand to subject donor hinds to repeated superovulation treatments within a single breeding season. This may be due largely to the necessity of performing surgical embryo recovery on donors and the desire to restrict the subsequent calving season of recipient hinds. However, the ability to successfully cryopreserve deer embryos and a possible increase
Ž
in the international trade of frozen embryos in preference to livestock Dixon et al.,
.
1991 may see a trend towards repeated superovulation and embryo recovery from elite hinds to maximise ova recoveries within a short timeframe. The present study demon-strates that superovulation can be performed over a 7–8 month period, although application in MOET may still be limited by other factors such as ova viability, stag fertility, embryo recovery technique, and individual responsiveness.
A question remains as to the effects of repeated exposure to oFSH on subsequent ovulatory responses. The greatest responses for F wapiti hybrids were observed during1 the first two treatment sessions, indicating possible desensitisation to oFSH as the trial progressed. This was not apparent for the other genotypes, although their overall ovulation rates were generally low throughout the study. A progressive reduction in ovulatory response to repeated treatment with eCG has been documented for sheep,
Ž .
cattle, and goats Pigon et al., 1960; Jainudeen et al., 1966; Baril et al., 1992 .
Ž .
Furthermore, Roy et al. 1999 demonstrated that the frequent use of eCG resulted in a marked humoral immune response in goats that is correlated with reduced ovarian activity. We considered the possibility that ovine FSH could be antigenic in cervids when administered with high frequency. However, our failure to demonstrate any antibodies to oFSH over the course of the trial indicates that the reduced ovulation rates for wapiti hybrid hinds were due to factors other than humoral antigenicity of the foreign FSH protein.
Acknowledgements
( ) G.W. Asher et al.rAnimal Reproduction Science 59 2000 61–70 70
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